SAG (Sensitive to Apoptosis Gone), also known as RBX2IROC2, or RNF7 (RING finger protein 7), is a stress-responsive component of SCF (Skp1, Cullins, F-box proteins) E3 ubiquitin ligase. Our previous work has shown that SAG promotes cell proliferation and Inhibits apoptosis under stressed conditions. The role of SAG in regulation of angiogenesis and carcinogenesis is, however, largely unknown. Our long-range goal is to achieve cancer therapy through inhibition of angiogenesis and tumorigenesis by targeting SAG. Our strong preliminary data showed that SAG knockout causes mouse embryonic lethality at E1O.5-12.5, largely due to the failure in vasculogenesis and angiogenesis. Embryoid bodies derived from SAG-null embryonic stem (ES) cells are unable to differentiate into endothelial cells to form the interior of blood islands, which is associated with the failure in VEGF induction and in RAS/ERK activation. The teratomas derived from SAG-null ES cells are smaller in size with reduced density of blood vessels and reduced rate of proliferation. We also found that transgenic expression of SAG accelerates the growth of mouse skin tumors induced by chemical carcinogens, whereas SAG knock-down by siRNA inhibits the growth of human cancer cells both in vitro and in vivo. The objectives of this application are 1) to define the role of SAG in angiogenesis and 2) to elucidate its mechanism of action, using mouse ES cells arid human cancer cells. The central hypothesis is that SAG promotes angiogenesis by inducing degradation of 1KB to activate NFKB and degradation of neurofibromin, a NF-1 gene product, to activate RAS. SAG deletion causes the accumulation of 1) lKBa to inactivate NFKB, and 2) neurofibromin to inactivate RAS, resulting in inhibition of angiogenesis. The specific aims to test the hypothesis are 1) to define the role of SAG in regulation of angiogenesis in mouse ES cells and in human cancer cells and 2) to define the mechanism of action by which SAG regulates endothelial differentiation of embryoid bodies. We expect that successful completion of this proposed study will demonstrate that blockade of SAG inhibits angiogenesis through NFKB inactivation by accumulated 1KB and RAS inactivation by accumulated neurofibromin. Targeting SAG in cancer cells, via siRNA silencing or small molecule inhibitors, would, therefore, be a valid antianglogenesis therapy.